3d islet formation from endocrine progenitor cells

Abstract

The present disclosure relates to a method for the generation of cells of the pancreatic lineage, for example pancreatic islet-like cell aggregates comprising pancreatic β-cells, which method comprises the steps of providing a single cell suspension of a population of endocrine progenitor (EP) cells, allowing said EP cells in single cell suspension to form 3D structures and culturing said cells under conditions permissive of differentiation into pancreatic monohormonal β-cells. The present disclosure also relates to pancreatic islet-like cell aggregates obtainable by said method as well as to medical uses thereof.

1 . Method for the generation of pancreatic islet-like cell aggregates in vitro, comprising the steps of
 i) providing a population comprising endocrine progenitor (EP) cells, characterized by the expression of NKX6.1 and NEUROD1 , wherein the population is an adherent culture of EP cells on a 2D substrate;   ii) providing a single cell suspension of said population of EP cells;   iii) allowing said population of EP cells in single cell suspension to form 3D structures;   iv) culturing said population of EP cells in the form of 3D structures in 3D culture conditions permissive of differentiation into pancreatic monohormonal β-cells to provide pancreatic islet-like cell aggregates; and   v) thereby generating pancreatic islet-like cell aggregates comprising monohormonal β-cells,   wherein said pancreatic islet-like cell aggregates comprise at most approximately 2% proliferating cells and wherein said pancreatic islet-like cell aggregates comprise at least approximately 40% monohormonal β-cells.   
     
     
         2 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein said 2D substrate comprises one or more components selected from the group consisting of laminins (LN) and fragments thereof, vitronectin and fragments thereof, fibronectin and fragments thereof, collagen and fragments thereof, gelatin and fragments thereof, functionalized silk (FN silk) and Matrigel. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein said pancreatic islet-like cell aggregates generated in step v) comprise approximately from 7 to 25% monohormonal α-cells. 
     
     
         7 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein said pancreatic islet-like cell aggregates generated in step v) comprise at most approximately 1% proliferating cells. 
     
     
         8 . (canceled) 
     
     
         9 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein step ii) is performed prior to culturing the cells in a medium permissive of differentiation into pancreatic monohormonal β-cells. 
     
     
         10 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein said formation of 3D structures in step iii) is spontaneous formation of 3D structures or wherein said formation of 3D structures in step iii) is forced or aided formation of 3D structures. 
     
     
         11 . (canceled) 
     
     
         12 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein step iv) comprises culturing EP for approximately 2 weeks or longer. 
     
     
         13 . (canceled) 
     
     
         14 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein the monohormonal β-cells generated in step v) have the ability to express C-peptide upon glucose stimulation. 
     
     
         15 . (canceled) 
     
     
         16 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein said pancreatic islet-like cell aggregates generated in step v) comprise at least 40% monohormonal β-cells; 7-25% monohormonal α-cells and less than 2% proliferating cells. 
     
     
         17 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein prior to step i) the method comprises the steps a−1)-c−1) and the steps a) and c) of
 a−1) providing a cell population of primitive gut tube cells characterized by expression of HNF1β and/or HNF4α; 
 b−1) culturing said cell population of primitive gut tube cells for no more than approximately 54 hours under conditions permissive of differentiation into posterior foregut cells; 
 c−1) thereby generating a population of posterior foregut cells characterized by the expression of PDX1. 
 a) providing the cell population of posterior foregut cells generated in step c−1) characterized by expression of PDX1; 
 b) culturing said cell population of posterior foregut cells for no more than approximately 78 hours under conditions permissive of differentiation into pancreatic progenitor cells; and 
 c) thereby generating a cell population of pancreatic progenitor cells characterized by expression of both PDX1 and NKX6.1. 
 
     
     
         18 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 17 , wherein further comprising, after the steps a)-c), the steps a+1)-c+1) of:
 a+1) providing a cell population of pancreatic progenitor cells generated in step c;   b+1) culturing said cell population of pancreatic progenitor cells under conditions permissive of differentiation into endocrine progenitor cells; and   c+1) thereby generating a population of endocrine progenitor cells characterized by expression of NKX6.1 and NEUROD1.   
     
     
         19 - 22 . (canceled) 
     
     
         23 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 18 , wherein in step b+1) said cell population of pancreatic progenitor cells is cultured for approximately from 3 to 5 days. 
     
     
         24 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 18  wherein the cells are cultured on a 2D substrate during steps a−1) to c+1). 
     
     
         25 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein the cells are not transferred from culture on a 2D substrate to culture on a 3D substrate prior to exhibiting expression of markers characteristic of endocrine progenitor cells. 
     
     
         26 . Method for the generation of pancreatic islet-like cell aggregates in vitro according to  claim 1 , wherein the EP cell population in step i) is derived from a culture of pluripotent stem cells. 
     
     
         27 - 30 . (canceled) 
     
     
         31 . Isolated pancreatic islet-like cell aggregates or population of pancreatic islet-like cell aggregates obtainable by the method according to  claim 1 , wherein said pancreatic islet-like cell aggregates comprise at least 40% monohormonal β-cells; approximately from 15 to 20% monohormonal α-cells and less than approximately 2% proliferating cells. 
     
     
         32 . (canceled) 
     
     
         33 . Isolated pancreatic islet-like cell aggregates or isolated population of pancreatic islet-like cell aggregates according to  claim 31 , wherein approximately from 40 to 50% of the total cell population are monohormonal β-cells characterized by the expression of insulin. 
     
     
         34 - 36 . (canceled) 
     
     
         38 . Isolated pancreatic islet-like cell aggregates or isolated population of pancreatic islet-like cell aggregates according to  claim 31 , wherein said pancreatic islet-like cell aggregates comprise from 7 to 20monohormonal α-cells. 
     
     
         38 - 47 . (canceled) 
     
     
         48 . Method of treatment of diabetes a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the isolated pancreatic islet-like cell aggregates or isolated population of pancreatic islet-like cell aggregates according to  claim 31 . 
     
     
         49 . Method of treatment of diabetes in a patient in need thereof, comprising the steps of generating isolated pancreatic islet-like cell aggregates according to the method as defined in  claim 1 ; and
 administering to said patient a therapeutically effective amount of said islet-like cell aggregates.   
     
     
         50 . (canceled)